This invention relates to oilseed plants, and more particularly to plants containing a nucleic acid encoding a cytosolic acetyl coA-carboxylase (ACCase) enzyme.
Acetyl-CoA carboxylase [ACCase; EC 6.4.1.2] catalyzes the first committed step in fatty acid biosynthesis by converting acetyl-CoA to malonyl-CoA. In plants, a multisubunit (MS) form and a multifunctional (MF) form of ACCase have been identified. The MS form is composed of dissociable subunits of different sizes, including a biotin carboxyl carrier protein (BCCP), xcex1- and xcex2-carboxyltransferases (xcex1-CT and xcex2-CT, respectively), and a biotin carboxylase (BC). The MS form is present in plastids of dicotyledenous and of non-Gramineae monocotyledenous plants and is primarily involved in the biosynthesis of fatty acids.
The MF form of a plant ACCase is similar to mammalian ACCase (and is sometimes designated xe2x80x9ceukaryoticxe2x80x9d or xe2x80x9ccytosolicxe2x80x9d ACCase), in that it is a MF polypeptide with a molecular weight of more than 200 kDa. The MF form of ACCase from plants contains BCCP, BC, xcex1-CT and xcex2-CT functional domains in a single polypeptide. MF ACCase is most likely present in the cytosol of all plant species and in the chloroplasts of Gramineae plants. Plant MF ACCase is involved in the biosynthesis of very long chain fatty acids, flavonoids, and in the malonation of amino acids and aminocyclopropane-1-carboxylate (a precursor to ethylene).
Antisense nucleic acids against an ME ACCase have been introduced into Brassica napus (White et al., 1998, in Adv. in Plant Lipid Res., pp. 62-66, eds., Sxc3xa1nchez, J., Cerdxc3xa1-Olmedo, E. and Martinez-Horce, E., Universidad De Sevilla, Spain) and an Arabidopsis genomic DNA encoding an MF ACCase under the control of a napin seed-specific promoter and linked to a small subunit (ss) Rubisco transit peptide was introduced into B. napus (Roesler et al., 1997, Plant Physiol., 113:75-81; U.S. Pat. No. 5,925,805).
Plants have been engineered to express a nucleic acid encoding an MF acetyl coA-carboxylase (ACCase), hereinafter referred to as cytosolic ACCase. Oil content was significantly increased in plants containing the cytosolic ACCase coding sequences.
In general, the invention feature plants containing a nucleic acid construct carrying a nucleic acid encoding a cytosolic ACCase operably linked to a promoter and lacking a transit peptide. This plant produces seeds that exhibit a statistically significant increase in oil content as compared to seeds produced by a corresponding plant lacking such a construct.
The invention additionally features plants containing a nucleic acid construct carrying a nucleic acid encoding a cytosolic ACCase lacking introns operably linked to a promoter. This plant produces seeds that exhibit a statistically significant increase in oil content as compared to seeds produced by a corresponding plant lacking such a construct.
The invention also features methods of producing a transgenic plant. This method includes selecting progeny transgenic plants of a plant containing a nucleic acid construct carrying a nucleic acid encoding a cytosolic ACCase operably linked to a promoter. Following at least one generation of selection, one or more of the progeny transgenic plants produce seeds exhibiting a statistically significant increase in oil content as compared to seeds produced by a corresponding plant lacking such a construct.
The invention further features methods of producing a plant by introducing a construct carrying a nucleic acid encoding a cytosolic ACCase operably linked to a promoter into one or more plants. Progeny of these plants, following at least one generation of selection, produce seeds that exhibit a statistically significant increase in oil content when compared to seeds produced by a corresponding plant lacking such a construct.
Yet another feature of the invention are methods of increasing the oil content in seeds by creating a plant containing a nucleic acid construct carrying a gene encoding a cytosolic ACCase operably linked to a promoter, and selecting progeny of the plant that exhibit a statistically significant increase in oil content in seeds as compared to seeds produced by a corresponding plant lacking such a construct.
Additionally featured in the invention are seeds produced by the above-described plants, and progeny of those plants, wherein the progeny produce seeds that exhibit a statistically significant increase in oil content when compared to seeds produced by the progeny of plants lacking such a construct.
Typically, the increase in oil content is from about 5% to about 25% on a dry weight basis. The above-described selection steps can include selecting progeny that contain the nucleic acid construct. Generally, soybean plants or Brassica plants, for example, Brassica napus, B. rapa, B. juncea, B. carinata, B. nigra and B. oleracea are useful in the invention.
Still yet another feature of the invention is a nucleic acid construct carrying a cytosolic ACCase coding sequence operably linked to a promoter but lacking a transit peptide and a nucleic acid construct carrying a cytosolic ACCase coding sequence lacking introns operably linked to a promoter.
A promoter included in a construct of the invention can be a cauliflower mosaic virus (CaMV) 35S promoter. Unless otherwise indicated, the ACCase constructs described herein may or may not include nucleic acid sequences encoding a transit peptide operably linked to the nucleic acid sequences encoding the cytosolic ACCase. An example of a transit peptide is a tobacco small subunit Rubisco transit peptide. In addition, a nucleic acid encoding a cytosolic ACCase can encode a plant cytosolic ACCase, for example, an alfalfa cytosolic ACCase. Further and unless otherwise indicated, a nucleic acid encoding the ACCase can lack introns.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The materials methods, and examples are illustrative only and not intended to be limiting. Suitable methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.